Shoot cultures of rare Balkan hyperaccumulating species Alyssum markgrafii were subjected to high nickel concentrations of 1, 3, 5, and 8 mM. The effects of graded nickel concentrations on toxicity, pigments, and several components of plant antioxidative defense system were characterized. Toxic effects of excess nickel were observed through slower growth and biomass decrease, together with increased reactive oxygen species (ROS) production and lipid peroxidation. Nickel exposure decreased chlorophyll a, b, total chlorophyll as well as carotenoid concentration. Addition of sodium benzoate, potent ROS scavenger, showed concentration-dependent disturbing effect on nickel hyperaccumulation, lowering the content of accumulated nickel in A. markgrafii shoots. General reducing power represented by low molecular weight antioxidants and phenols was inversely correlated with nickel concentration. Among the investigated antioxidative enzymes, POD seems to play important role in ROS level regulation upon excessive nickel in medium.